Cosmetic or pharmaceutical and particularly dermatological, composition containing an extract of tephrosia, particularly Tephrosia purpurea

ABSTRACT

The present invention relates to a cosmetic or a pharmaceutical composition, in particular a dermatological composition. The composition is characterized in that it comprises a cosmetically or pharmaceutically, and in particular dermatologically, effective quantity of an extract of a plant of genus Tephrosia, in particular an extract of the species Tephrosia purpurea. According to the invention, it has been discovered that the extract of the plant of genus Tephrosia is useful in preparing a cosmetic, pharmaceutical, and in particular dermatological composition, presenting body-slimming, pigmenting, anti-aging, or anti-inflammatory activity.

The present invention relates essentially to the use of an extract ofTephrosia, in particular Tephrosia purpurea for preparing apharmaceutical composition, in particular a dermatological composition,or as a cosmetic agent, and also to a method of cosmetic treatmentconstituting an application thereof.

Plants of the genus Tephrosia are subtropical plants widely distributed,in particular in India and in Sri Lanka.

A species of the genus Tephrosia that is particularly preferred in thecontext of the invention is the plant of the species Tephrosia purpurea.It is a perennial plant having a height of 30 cm to 100 cm, giving 4 to6 seeds per pod, and having red flowers. The species Tephrosia purpureais particularly known in India and it is known essentially in Ayurvedicmedicine, i.e. in traditional Indian medicine. Various parts have beenused to treat a variety of diseases: bronchial, liver, and kidneydiseases, and blood purification (see article by Pandey in Quart. J.Crude Drug Res. 13 (1975), pp. 65-68). Seeds of Tephrosia purpurea havealso been used for hypoglycemic activity (see article by Rahman et al.,in Indian J. Med. Res. 81, April 1985, pp. 418-421). In vitroantimicrobial activity of Tephrosia purpurea is also known (see articleby Agarwal et al. in Indian J. Physiol. Pharmac., Vol. 31, No. 4,October-December 1987, pp. 284-286) using a root extract in 90% alcoholin a Soxhlet apparatus.

After intensive research on plants of the genus Tephrosia, and inparticular of the species Tephrosia purpurea, the present inventors havediscovered, unexpectedly, that extracts, and in particular extracts ofthe seeds, of the plant Tephrosia, in particular Tephrosia purpurea,present powerful stimulation activity for the enzyme adenylate cyclase.It is known that this enzyme transforms ATP into AMPc and pyrophosphate.Because of this activity, extracts of the genus Tephrosia, in particularof the species Tephrosia purpurea, are valuable for an application incosmetics or in pharmacy, in particular in dermatology, in body-slimmingactivity, in anti-inflammatory activity, in pigmenting activity byacting on melanocytes, and in anti-aging activity.

Thus, in a first aspect, the present invention covers the use of anextract of a plant of the genus Tephrosia, in particular the speciesTephrosia purpurea, as an active substance for preparing apharmaceutical composition, in particular a dermatological composition,presenting activity in stimulating the enzyme adenylate cyclase,body-slimming activity, anti-inflammatory activity, pigmenting activity,and anti-aging activity, in particular when applied topically,optionally in a medium, vehicle, or excipient that is pharmacologically,and in particular dermatologically, acceptable.

The invention also covers the use of an extract of a plant of the genusTephrosia, in particular of the species Tephrosia purpurea, as acosmetic agent for stimulating the enzyme adenylate cyclase, to obtain abody-slimming effect, to obtain a pigmenting effect, or to obtain ananti-aging effect, in particular when applied topically.

In an advantageous embodiment, the above-specified extract is a seedextract of a Tephrosia plant, in particular a seed extract of thespecies Tephrosia purpurea.

It should be observed that the plant Tephrosia purpurea is also known,in particular according to the Napralert® database under the followingsynonymous names: Cracca purpurea, Galeduba lanceaefolia, Galedubapurpurea, Galepa tinctoria, Tephrosia galegoides, Tephrosia indigofera,Tephrosia lobata, and Tephrosia maxima, all of which are naturallycovered by the present invention.

In an advantageous embodiment, this seed extract is a hydroalcoholicextract with a C₁ -C₆ alcohol that is linear or cyclic branched. Aparticularly preferred alcohol is methanol or ethanol. The relativeproportions of water and alcohol may range over wide limits.Nevertheless, substantially equal volume proportions are preferred.

In a second aspect, the present invention also covers a method ofcosmetic treatment for obtaining stimulation of the enzyme adenylatecyclase to obtain a body-slimming effect, to obtain a pigmenting effect,or an anti-aging effect, characterized in that it comprises topicalapplication on the areas concerned of a patient, in particular theepidermis or the hair, of a cosmetically effective quantity of anextract of a plant of the genus Tephrosia, in particular the speciesTephrosia purpurea, optionally in a cosmetically acceptable excipient,medium, or vehicle.

Advantageously, in any of the preceding aspects, 0.001% to 5%, andpreferably 0.01% to 5%, by weight of extract is used relative to thetotal weight of the composition.

Other objects and advantages of the invention appear clearly in thelight of the following explanatory description made with reference tovarious embodiments of the invention given purely by way of illustrationand that do not in any way limit the scope of the invention. Unlessspecified otherwise, percentages given in the description and the claimsare given by weight.

EXAMPLE 1

Making a plant extract from Tephrosia purpurea

1000 grams (g) of Tephrosia purpurea seeds purchased commercially weretaken and ground and passed through a 1 mm screen. The screening wassteeped for 24 h in 5 liters (l) of hydroalcoholic extraction solventconstituted in this case by a 50/50 V/V methanol-water mixture.

It was refluxed for 1 h.

It was then filtered on paper (pressure could have been used). It waswashed in cold methanol.

The alcohol and the water were then vacuum evaporated off until they hadcompletely disappeared from the filtrate.

The residue was weighed, giving 80.11 g of extract of non-uniformappearance comprising a clear crystalloid and a dark paste.

The raw extract contained about 50% dry matter and 50% residualsolvents.

It was suitable for use as such, or it could have been further purifiedby conventional methods well known to the person skilled in the art.

EXAMPLE 2

2,365 kg of commercially available seeds of the plant Tephrosia purpureawere ground and screened with a 1 mm screen, and then degreased withpetroleum ether.

The ground and degreased seeds were then subjected to extraction for 2hours in a Soxhlet apparatus with about 10 l of ethanol.

After the solvent had been evaporated off, a residue was obtained in theform of a brownish gum weighing about 58 g.

EXAMPLE 3

Use of Tephrosia purpurea extract to show stimulation of adenylatecyclase activity

Certain substances act as intracellular messengers, and as a result theyare essential constituent substances of cells that adapt the functioningof cells to the requirements of the organism. Such messengers include3',5' adenosine which is a cyclic monophosphate (AMPc). This molecule ismetabolized from adenosine 3',5'-triphosphate by an enzyme calledadenylate cyclase.

The theoretical reaction is as follows: ##EQU1## Theory

Cell membranes are initially obtained by culturing to confluencefibroblasts of the 3T3 F442A line commercially available from FlowLaboratories.

These fibroblast cell membranes which constitute an enzyme source, areincubated in a reaction medium in the presence of ATP at 37° C. for apredefined length of time.

The resulting AMPc is measured by radioimmunoassay. Taking the basalactivity, i.e. activity without an effector, as being 100%, it ispossible to determine the influence of an effector on the enzyme system.If the activity is greater than 100%, then there is an activator effectof adenylate cyclase, and if the activity is less than 100%, then on thecontrary an inhibitor effect is demonstrated.

Implementation

Cell membranes are purified from the fibroblast in culture.

In this context, it should be observed that purification of cellmembranes releases numerous enzymes into the assay medium. Two of theseenzymes are undesirable in implementing the test. They comprise firstlyNa⁺, K⁺ ATPase which transform ATP into adenosine diphosphate, andsecondly phosphodiesterase 3',5'-AMPc which degrades cyclic AMP intoAMP. Thus, in order to obtain reliable results from the test, it isessential to inhibit those two enzymes. The first is inhibited by meansof ouabain, and the second by means of theophylline.

The reaction medium is thus constituted by:

50 μl of a 5 mM solution of adenosine 3',5'-triphosphate (ATP) assubstrate;

100 μl of a 20 mM solution of ouabain as an inhibitor of ATPase;

100 μl of a 20 mM solution of theophylline as an inhibitor ofphosphodiesterase; and

100 μl of a solution containing 0.05 g/l dry matter of the effector.

After incubation, the reaction is stopped by heating for 10 minutes to100° C., centrifuging, and recovering the supernatent to measure theAMPc by radioimmunoassay (kit reference 1117 from the French companyImmunotech).

Results

The results of tests are given in the table below:

                  TABLE 1                                                         ______________________________________                                                                      Tephrosia                                                           Positive  purpurea                                                            reference =                                                                             at 0.05 g/l                                              Basal      forskoline                                                                              dry extract                                              activity   at 0.05 g/l                                                                             from Example 2                                  ______________________________________                                        % activity                                                                             100        225       185                                             ______________________________________                                    

It can be seen very clearly from the above results that the extract fromthe plant Tephrosia purpurea possesses large significant activity instimulating the enzyme adenylate cyclase. This activity is relativelyclose to that of the positive reference as constituted by forskoline,even though the plant extract does not contain that molecule.

Thus, Tephrosia extracts, in particular Tephrosia purpurea extracts,constitute an interesting alternative for forskoline in its use inpreparing cosmetic or pharmaceutical compositions.

Applications of the present invention are, in particular, those whichstem from biological processes in which AMPc plays a role. Of suchapplications, those preferred in the invention are preparing slimmingcompositions, pigmenting compositions for the skin or the hair,anti-inflammatory compositions, and compositions for opposing theeffects of aging.

EXAMPLE 4 Demonstrating lipolytic activity in the composition of theinvention

Evaluating the action of the composition of the invention on adipocytesin culture

It was decided to evaluate the effectiveness of compositions of theinvention as lipolytic agents on a line of mouse pre-adipocytes, such asa 3T3 F442A line commercially available from Flow Laboratories, whichhave been selected for their ability to convert into adipocytes ifculture conditions make that possible.

(In application of the method of H. Green & C. Kehinde, Cell, 1 (1974),113.)

This line constitutes a model for studying in vitro adipocytedifferentiation while providing the possibility, when the adipocytephenotype is achieved, of studying extracellular regulation of cellfunctioning in accordance with the invention. This differentiation, andmodulation thereof, are accompanied by a certain number of morphologicaland biochemical changes, and in particular biochemical changesconcerning the release of glycerol during lipolysis.

It is thus convenient to verify the effectiveness of compounds to betested by measuring the activity of the enzyme glycerol-3-phosphatedehydrogenase (written G3PDH) which is involved in the following lipidgenerating reaction: ##STR1##

Thus, enzyme activity can be measured using the method of assaying therelease NADH by the method described by J. Pairault et al. in Proc. Nat.Acad. Sci., Volume 6 (1979), pp. 5138-42.

These experiments were performed as follows:

1) Culture conditions

The pre-adipocytes were seeded in 35 mm diameter Petri dishes (20,000cells/dish) in the presence of Dulbecco's Modified Eagle Medium (DMEM)containing 5% calf serum, plus 5% fetal calf serum.

The medium was renewed twice with DMEM medium plus 10% fetal calf serum.

Under such conditions, the culture reached confluence in 1 week (D=D0),at which stage adipocyte differentiation was activated by adding insulinat a concentration of 1% by weight of culture medium. There were alsotwo changes of the DMEM culture medium plus 10% fetal calf serum and 1%insulin.

The cells presented an advanced differentiated state 1 week afterconfluence (D=D7).

2) Treatment-viability

The cells were treated with the substances to be tested at stage D7.

The treatment consisted in replacing the culture medium either by mediumonly as a reference, or else by the same medium but containing thesubstance to be tested at various concentrations.

To assay G3PDH activity, the culture medium was recovered 7 days aftertreatment (D=D14).

3) Assaying G3PDH enzyme activity on day D14

The monolayer of cells was recovered by scraping and it was vigorouslyhomogenized in a 25 mM TRIS-HCl buffer, pH 7.4, with 1 mM EDTA at 4° C.G3PDH activity was assayed on the cell particle supernatent aftercentrifuging.

The assay, performed using the above-mentioned Pairault assay method,measured G3PDH enzyme activity per milligram of protein. The proteincontent was determined by the protein assay method described by J. Lowryin Biol. Chem., 193 (1951), pp. 265-75, with enzyme activity beingexpressed in nanomoles/min/mg of protein.

Three tests were performed per sample or per reference.

The following substances were tested:

a) a first reference substance for which the DMEM medium had addedthereto 7.5 μl of ethanol for 6 ml of culture medium;

b) the same medium but having 60 μl of DMSO (dimethylsulfoxide) added to6 ml of culture medium;

c) the same medium but having added thereto an extract of Coleusforskohlii containing forskoline, which is commercially available, at aconcentration of 0,025 g/l dry matter diluted in 7.5 μl of ethanol for 6ml of culture medium;

d) a substance in which the above DMEM medium had added thereto 0.2 g/ldry extract of Tephrosia purpurea as obtained from Example 2, diluted in60 μl of DMSO for 6 ml of culture medium;

e) a substance in which the DMEM medium had added thereto 0.1 g/l dryextract of Tephrosia purpurea as obtained from Example 2, diluted in 60μl of DMSO for 6 ml of culture medium; and

f) a substance in which the DMEM medium had added thereto 0.05 g/l dryextract of Tephrosia purpurea as obtained from Example 2, diluted in 60μl of DMSO for 6 ml of culture medium.

The results obtained are given in Table 2 below:

                  TABLE 2                                                         ______________________________________                                                       G3PDH activity expressed                                                      as nanomoles of NADH                                                          released per min/ml of                                         Substance under test                                                                         protein                                                        ______________________________________                                        Ethanol reference                                                                            382.68                                                         DMSO reference 392.36                                                         Coleus forskohlii                                                                            176.48                                                         Tephrosia (0.2 g/l)                                                                          155.23                                                         Tephrosia (0.1 g/l)                                                                          353.03                                                         Tephrosia (0.05 g/l)                                                                         369.64                                                         ______________________________________                                    

It can be seen very clearly from the results of Table 2 above that theextract of the plant Tephrosia purpurea possesses considerablesignificant activity in inhibiting the activity of G3PDH, as shown bythe small quantity of NADH released. In addition, from a concentration0.02 g/l, this activity is similar or slightly greater than the activityobtained with the extract of Coleus forskohlii which containsforskoline, known for its activity in stimulating adenylate cyclase andits effect on lipolyses.

It can be advantageous to inhibit G3PDH activity using a substance thatdoes not contain forskoline, thus providing an alternative of interestto forskoline in its use for preparing cosmetic or pharmaceuticalcompositions for the activities mentioned above.

The present invention is described below with reference to variousexamples of formulations for cosmetic or pharmaceutical compositions, inparticular dermatological compositions.

EXAMPLE 5

Body slimming cream

Tephrosia purpurea seed extract of Example 1 1%

Excipient for cream q.s.p. 100%

This cream is applied at least once a day for 2 to 3 weeks to areas ofthe body having fatty substances, until the desired slimming effect isobtained.

EXAMPLE 6

Pigmenting cosmetic composition

This composition comprises the following ingredients:

Tephrosia purpurea seed extract of Example 1 0.5%

Ordinary cosmetic gel excipient q.s.p. 100%

The gel is applied to areas of the skin that are to be pigmented untilthe desired effect is obtained.

EXAMPLE 7

Anti-inflammatory pharmaceutical composition, in particular adermatological composition

This composition comprises the following ingredients:

Tephrosia purpurea seed extract of Example 1 0.2%

Pharmaceutically acceptable excipient q.s.p. 100%

EXAMPLE 8

Anti-aging cosmetic composition

This composition comprises the following ingredients:

Tephrosia purpurea seed extract of Example 1 0.1%

Usual cosmetic excipient q.s.p. 100%

This composition is applied to areas of the skin where an anti-agingeffect is sought after, and it is applied for a sufficient period oftime, generally of the order of several weeks to several monthsdepending on the subject.

We claim:
 1. A method of performing a treatment of body zones selectedfrom the epidermis and the hair, comprising stimulating the enzymeadenylate cyclase by applying on the body zones a stimulating effectiveamount of an extract of a plant of the genus Tephrosia.
 2. The method ofclaim 1, wherein said plant is of the species Tephrosia purpurea.
 3. Themethod of claim 1, wherein said extract is administered as a compositionhaving a concentration of said extract ranging between 0.001% and 5% byweight with respect to the total weight of the composition.
 4. Themethod of claim 1, wherein said extract is an extract obtained by use ofan extraction solvent selected from the group consisting of a C₁ -C₆alcohol and a C₁ -C₆ hydroalcohol.
 5. The method of claim 1, wherein theextract is selected from the group consisting of methanol and ethanol.6. The method of claim 1, wherein said extract is a seed extract.
 7. Themethod of claim 1, wherein said extract is a seed extract of the speciesTephrosia purpurea.
 8. The method of claim 1, wherein said extract is a50/50 V/V methanol-water solvent extract of Tephrosia purpurea seeds. 9.The method of claim 1, wherein said extract is an ethanol extract of theplant Tephrosia purpurea.
 10. A method for providing a cosmetic effectfor a person, comprising delivering to said person an effective amountfor said cosmetic effect of an extract of a plant of the genusTephrosia.
 11. The method of claim 10, wherein said cosmetic effect isfor slimming.
 12. The method of claim 11, wherein said extract isadministered as a composition having a concentration of said extractranging between 0.001% to 5% by weight with respect to the total weightof the composition.
 13. The method of claim 10, wherein said cosmeticeffect is for pigmenting.
 14. The method of claim 10, wherein saidcosmetic effect is for reducing signs of aging.
 15. The method of claim10, wherein said cosmetic effect is anti-inflammatory.
 16. The method ofclaim 10, wherein said plant is of the species Tephrosia purpurea. 17.The method of claim 10, wherein said extract is administered as acomposition having a concentration of said extract ranging between0.001% to 5% by weight with respect to the total weight of thecomposition.
 18. The method of claim 10, wherein said extract is anextract obtained by use of an extraction solvent selected from the groupconsisting of a C₁ -C₆ alcohol and a C₁ -C₆ hydroalcohol.
 19. The methodof claim 18, wherein the solvent is selected from the group consistingof methanol and ethanol.
 20. The method of claim 10, wherein saidextract is a seed extract.
 21. The method of claim 10, wherein saidextract is a seed extract of the species Tephrosia purpurea.
 22. Themethod of claim 10, wherein said extract is a 50/50 V/V methanol-watersolvent extract of Tephrosia purpurea seeds.
 23. The method of claim 10,wherein said plant is an ethanol extract of the plant Tephrosiapurpurea.
 24. The method of claim 10, comprising applying said extracttopically on body zones selected from the epidermis and the hair.
 25. Amethod for performing a pharmaceutical treatment selected from the groupconsisting of a slimming treatment and an anti-inflammatory treatment ona subject, comprising delivering to the subject an effective amount forsaid pharmaceutical treatment of an extract of a plant of the genusTephrosia.
 26. The method of claim 25, wherein said plant is of thespecies Tephrosia purpurea.
 27. The method of claim 26, wherein saidextract is administered as a composition having a concentration of saidextract ranging between 0.001% and 5% by weight with respect to thetotal weight of the composition.
 28. The method of claim 25, whereinsaid extract is administered as a composition having a concentration ofsaid extract ranging between 0.001% and 5% by weight with respect to thetotal weight of the composition.
 29. The method of claim 25, whereinsaid extract is an extract obtained by the use of an extraction solventselected from the group consisting of a C₁ -C₆ alcohol and a C₁ -C₆hydroalcohol.
 30. The method of claim 29, wherein the solvent isselected from the group consisting of methanol and ethanol.
 31. Themethod of claim 25, wherein said extract is a seed extract.
 32. Themethod of claim 25, wherein said extract is a seed extract of thespecies Tephrosia purpurea.
 33. The method of claim 25, wherein saidextract is a 50/50 V/V methanol-water solvent extract of Tephrosiapurpurea seeds.
 34. The method of claim 25, wherein said plant is anethanol extract of the plant Tephrosia purpurea.
 35. The method of claim25, comprising applying said extract topical on body zones selected fromthe epidermis and the hair.